Dexamethasone increases αvβ3 integrin expression and affinity through a calcineurin/NFAT pathway

Abstract

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p<0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6 days of treatment and then an additional 10 days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1 day of DEX treatment to increase levels for 4 days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7 h from 22.5 h in control cultures (p<0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway.

NFATc1 activity regulates the expression of myocilin induced by dexamethasone

“…Recent studies examining how DEX regulates the expression of proteins in the TM show that MYOC is not the only protein up regulated as a result of a secondary glucocorticoid response. The β3 integrin subunit in HTM cells is also up regulated by DEX (Faralli et al, 2013) and this study showed that a calcineurin/NFAT (nuclear factor of activated T-cells) pathway may be involved. Calcineurin is a serine/threonine phosphatase that is modulated by intracellular calcium levels.…”

“…This increase was inhibited with cyclosporin A (CsA) (Faralli et al, 2013), an immunosuppressant that forms a complex with cyclophilin leading to an inhibition of calcineurin phosphatase activity (Liu et al, 1991). To determine if CsA could also inhibit the DEX-induced increase in MYOC mRNA, we pre-treated HTM cells with 1 or 10 μM CsA for 1 h prior to the addition of DEX or EtOH.…”

“…Western blot analyses were performed as previously described (Faralli et al, 2013) with a few modifications. Briefly, HTM cells were lysed with lysis buffer (25 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1% NP-40, 0.25% deoxycholate, HALT phosphatase inhibitor cocktail and HALT protease inhibitor cocktail (Thermo Fisher Scientific)).…”

“…Relative quantification of the RT-qPCR data was performed according to Pfaffl (Pfaffl, 2001), using ITGB1 mRNA for normalization. We have determined from previous experiments that ITGB1 mRNA levels are unaffected by the treatments used in this study (Faralli et al, 2013). Statistical comparisons were done using the Student T-test and a p value < 0.05 was considered significant.…”

A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells

“…These co-signaling events could be the result of glucocorticoid-induced up-regulation and activation of αvβ3 integrins [67] and ECM proteins [28, 34]. Glucocorticoids have previously been shown to increase production and matrix deposition of unidentified laminins as well as thrombospondin-1, fibronectin and type IV collagen in HTM cells [68–70].…”